Many disease states are characterized by the uncontrolled proliferation and differentiation of cells. These disease states encompass a variety of cell types and maladies such as cancer, atherosclerosis, restenosis, arthritis, dermatitis, vein graft intimal hyperplasia, neointimal hyperplasia of vascular smooth muscle and psoriasis. The compounds can also be used to delay aging of the skin. The proliferation, differentiation and survival of cells are regulated by numerous extracellular signaling polypeptide growth factors, which have been implicated in these disease states. Some of the better characterized growth factors include the following: epidermal growth factor (EGF), fibroblast growth factors (FGFs), and platelet-derived growth factor (PDGF).
The effects of many growth factors are known to be mediated by high affinity receptor tyrosine kinases. The binding of growth factors to extracellular receptors activates intracellular tyrosine kinases that catalyze the phosphorylation of several tyrosines on intracellular protein substrates or the receptor (autophosphorylation). These phosphorylated tyrosines create high affinity binding sites for many secondary cellular proteins involved in signal transduction such as phosphatidylinositol 3-kinase (PI 3-kinase), phospholipase C-.gamma. (PLC-.gamma.), and ras-GTPase-activating protein (GAP), among others. These molecules contain homologous regions known as src homology 2 (SH2) domains that were first identified in src family protein tyrosine kinases (PTKs). SH2 domains confer high affinity interactions with specific phosphorylated tyrosine residues of the growth factor receptors. Further downstream signaling results in cellular proliferation. Thus, the blockade of these signal transduction pathways can be used in the treatment of proliferative diseases.
In particular, the binding of PDGF to cell surface receptors induces receptor dimerization, followed by autophosphorylation at multiple tyrosine residues, which initiates cytoplasmic signaling via secondary cellular proteins containing SH2 domains. Currently, more than twenty cytosolic proteins likely to be involved in signaling have been shown to contain SH2 domains. Of these, PI 3-kinase is an important member that interacts with many activated PTKs and is involved in both normal and oncogenic signal transduction. The role of PI 3-kinase in PDGF-mediated cell proliferation has been investigated by measuring the levels of DNA synthesis in NMuMG cells expressing wild-type versus those expressing mutant PDGF receptors. A significant increase in DNA synthesis was observed in cells expressing PDGF receptors that were specifically associated with PI 3-kinase. The important involvement of PI 3-kinase in cell motility was recently demonstrated by Wennstrom et al., Oncogene, 1994;9:651-60, who showed that in porcine aortic endothelial cells expressing the PDGF-.beta. receptor, membrane ruffling and chemotaxis transduced by the PDGF-.beta. receptor required PI 3-kinase binding.
PI 3-kinase is a heterodimeric enzyme and contains an 85 kDa (p85) noncatalytic subunit and a 110 kDa (p110) catalytic subunit. The p85 subunit has one src homology 3 (SH3) and two SH2 domains which bind to specific phosphorylated tyrosines on activated growth factor receptors. PI 3-kinase has been shown to interact specifically with the phosphorylated tyrosine 740 and tyrosine 751 residues of the PDGF-.beta. receptor.
Of the two SH2 domains (N- and C-terminal) of the p85 subunit of PI 3-kinase, the C-terminal SH2 domain like the full-length p85 distinguishes between the wild-type and a mutant PDGF receptor lacking the PI 3-kinase binding site. Thus, the C-terminal SH2 domain of the p85 subunit (p85 C-SH2) accounts for the high affinity and specificity of the binding of PI 3-kinase to the PDGF-.beta. receptor.